Protein arginine deiminase 4 inhibition is sufficient for the amelioration of collagen-induced arthritis.

Citrullination of joint proteins by the protein arginine deiminase (PAD) family of enzymes is recognized increasingly as a key process in the pathogenesis of rheumatoid arthritis. This present study was undertaken to explore the efficacy of a novel PAD4-selective inhibitor, GSK199, in the murine collagen-induced arthritis model of rheumatoid arthritis. Mice were dosed daily from the time of collagen immunization with GSK199. Efficacy was assessed against a wide range of end-points, including clinical disease scores, joint histology and immunohistochemistry, serum and joint citrulline levels and quantification of synovial autoantibodies using a proteomic array containing joint peptides. Administration of GSK199 at 30 mg/kg led to significant effects on arthritis, assessed both by global clinical disease activity and by histological analyses of synovial inflammation, pannus formation and damage to cartilage and bone. In addition, significant decreases in complement C3 deposition in both synovium and cartilage were observed robustly with GSK199 at 10 mg/kg. Neither the total levels of citrulline measurable in joint and serum, nor levels of circulating collagen antibodies, were affected significantly by treatment with GSK199 at any dose level. In contrast, a subset of serum antibodies reactive against citrullinated and non-citrullinated joint peptides were reduced with GSK199 treatment. These data extend our previous demonstration of efficacy with the pan-PAD inhibitor Cl-amidine and demonstrate robustly that PAD4 inhibition alone is sufficient to block murine arthritis clinical and histopathological end-points.

Smoking is not associated with autoantibody production in systemic lupus erythematosus patients, unaffected first-degree relatives, nor healthy controls.

OBJECTIVE: The objective of this paper is to examine whether smoking is associated with autoantibody production in systemic lupus erythematosus (SLE) patients, unaffected first-degree relatives (FDR) of individuals with SLE--a group at increased risk of developing SLE--or unaffected, unrelated controls. METHODS: Detailed demographic, environmental, clinical, and therapeutic information was collected by questionnaire on 1242 SLE patients, 981 FDRs, and 946 controls in the Lupus Family Registry and Repository; a blood sample was obtained. All sera were tested for multiple lupus autoantibodies by immunofluorescence and luminex bead-based assays. Generalized estimating equations, adjusting for age, gender, and ethnicity and accounting for correlation within families, were used to assess smoking status with the dichotomous outcome variables of positivity for SLE status, positivity of ANA by immunofluorescence (≥1:120), positivity for ≥1 autoantibody by the luminex assay, and positivity for each of the 11 autoantibodies. RESULTS: Current smoking was associated with being positive for ≥1 autoantibody (excluding ANA) (adjusted OR = 1.53, 95% CI 1.04-2.24) in our subjects with SLE. No association was observed in unaffected FDRs or healthy controls. Former smoking was associated with anti-Ro/SS-A60 in our unaffected FDRs. There was an increased association with anti-nRNP A seropositivity, as well as a decreased association with anti-nRNP 68 positivity, in current smokers in SLE subjects. CONCLUSIONS: No clear association between smoking status and individual autoantibodies was detected in SLE patients, unaffected FDRs, nor healthy controls within this collection. The association of smoking with SLE may therefore manifest its risk through mechanisms outside of autoantibody production, at least for the specificities tested.

Studies of Filipino patients with systemic lupus erythematosus: autoantibody profile of first-degree relatives.

This study surveyed the frequency of autoantibodies among un-affected first-degree relatives (FDRs) of Filipino systemic lupus erythematosus (SLE) patients compared with healthy un-related Filipino controls. The sensitivity, specificity and predictive value of the autoantibodies for SLE diagnosis were also assessed in this Filipino cohort. Filipino patients included in the University of Santo Tomas (UST) Lupus Database and un-affected FDRs were recruited. Healthy controls included those with no known personal or family history of autoimmune disease. The following autoantibodies were tested in all subjects: anti-nuclear antibody (ANA), anti-dsDNA, anti-Ro/SSA, anti-chromatin, anti-thyroid microsome, and anti-cardiolipin antibodies. Participants included 232 SLE patients, 546 FDRs, and 221 healthy controls. Median age of patients was 27 (range 8-66) years with median disease duration of 27.5 (range 1-292) months. Median age of FDRs was 42.0 (range 5-87) years. Compared with healthy controls, there were significantly more FDRs with positive ANA at titers 1 : 40 to 1 : 160 (p < 0.001) and 1 : 320 (p = 0.003), anti-Ro/SSA (4.94% versus 0.45%, p = 0.003), and anti-dsDNA ≥ 5.0 IU/ml (4.58% versus 1.36%, p = 0.031). ANA titer ≥1 : 160, anti-dsDNA, anti-Ro/SSA and anti-chromatin had the highest predictive value for SLE diagnosis. These findings reinforce the role of genetic influence in SLE risk among Filipinos, with a significant proportion of un-affected FDRs of SLE patients testing positive for autoantibodies compared with healthy Filipino controls. A longitudinal observational study in this same cohort will determine which proportion of these un-affected FDRs will evolve into clinical SLE disease in the future.

Supervised machine learning and logistic regression identifies novel epistatic risk factors with PTPN22 for rheumatoid arthritis.

Investigating genetic interactions (epistasis) has proven difficult despite the recent advances of both laboratory methods and statistical developments. With no 'best' statistical approach available, combining several analytical methods may be optimal for detecting epistatic interactions. Using a multi-stage analysis that incorporated supervised machine learning and methods of association testing, we investigated epistatic interactions with a well-established genetic factor (PTPN22 1858T) in a complex autoimmune disease (rheumatoid arthritis (RA)). Our analysis consisted of four principal stages: Stage I (data reduction)-identifying candidate chromosomal regions in 292 affected sibling pairs, by predicting PTPN22 concordance using multipoint identity-by-descent probabilities and a supervised machine learning algorithm (Random Forests); Stage II (extension analysis)-testing detailed genetic data within candidate chromosomal regions for epistasis with PTPN22 1858T in 677 cases and 750 controls using logistic regression; Stage III (replication analysis)-confirmation of epistatic interactions in 947 cases and 1756 controls; Stage IV (combined analysis)-a pooled analysis including all 1624 RA cases and 2506 control subjects for final estimates of effect size. A total of seven replicating epistatic interactions were identified. SNP variants within CDH13, MYO3A, CEP72 and near WFDC1 showed significant evidence for interaction with PTPN22, affecting susceptibility to RA.

The interrelationship of complement-activation fragments and angiogenesis-related factors in early pregnancy and their association with pre-eclampsia.

OBJECTIVE: To determine the interrelationships during early pregnancy of complement-activation fragments Bb, C3a and sC5b-9, and angiogenesis-related factors placental growth factor (PiGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), and their associations with pre-eclampsia. DESIGN: Prospective cohort study. SETTING: Denver complement study (June 2005-June 2008). POPULATION: A total of 668 pregnant women with singleton gestations, recruited between 10 and 15 weeks of gestation. METHODS: Using univariable and multivariable logistic regression analysis, concentrations of complement-activation fragments and angiogenesis-related factors were compared between 10 and 15 weeks of gestation in women who subsequently did or did not develop pre-eclampsia. Interrelationships between these variables were tested using the non-parametric Spearman rank correlation coefficient. MAIN OUTCOME MEASURE: Pre-eclampsia. The association of complement-activation fragments and angiogenesis-related factors with obesity was also examined. RESULTS: The mean (+/-SD) levels of complement Bb in early pregnancy among women who did and did not develop pre-eclampsia were 0.84 (+/-0.26) microg/ml and 0.69 (+/-0.2) microg/ml, respectively (P = 0.001). Concentrations of PiGF were significantly (P = 0.01) lower (31 +/- 12 pg/ml) in early pregnancy in the pre-eclamptic group of women, as compared with the normotensive group (39 +/- 32 pg/ml). The adjusted odds ratio (AOR) of Bb and PiGF were 2.1 (CI = 1.4-3.1, P < 0.0003) and 0.2 (CI = 0.07-0.7, P = 0.01), respectively. There was no significant difference in the levels of C3a, sC5b-9, sFlt-1 and sEng in early pregnancy among women who developed pre-eclampsia, compared with women who remained normotensive during pregnancy. Higher levels of Bb (P = 0.0001) and C3a (P = 0.03), and lower levels of sFlt-1 (P = 0.0002) and sEng (P = 0.0001) were found among women with obesity, compared with non-obese controls. No meaningful relationships were found between the complement-activation fragments and the angiogenesis-related factors. CONCLUSIONS: In this cohort during early pregnancy, increased concentrations of complement-activation factor Bb and lower concentrations of PiGF were associated with the development of pre-eclampsia later in pregnancy.

Complement activation pathways in murine immune complex-induced arthritis and in C3a and C5a generation in vitro.

The alternative pathway (AP) of complement alone is capable of mediating immune complex-induced arthritis in the collagen antibody-induced arthritis (CAIA) model in mice. Whether the classical pathway (CP) or lectin pathway (LP) alone can mediate CAIA is not known. Using mice genetically deficient in different complement components, our results reported herein establish that the CP and LP alone are each incapable of mediating CAIA. A lower level or absence of C3 and/or C5 activation by the CP may be possible explanations for the importance of the AP in CAIA and in many murine models of disease. In addition, other investigators have reported that CP C5 convertase activity is absent in mouse sera. To address these questions, we employed an in vitro system of adherent immunoglobulin (Ig)G-induced complement activation using plates coated with murine anti-collagen monoclonal antibody (mAb). These experiments used complement-deficient mouse sera and wild-type mouse or normal human sera under conditions inactivating either the CP (Ca(++) deficiency) or the AP (mAb inhibitory to factor B). Robust generation of both C3a and C5a by either the AP or CP alone were observed with both mouse and human sera, although there were some small differences between the species of sera. We conclude that neither the CP nor LP alone is capable of mediating CAIA in vivo and that mouse sera exhibits a high level of IgG-induced C5a generation in vitro through either the CP or AP.

Increased expression of activation-induced cytidine deaminase is associated with anti-CCP and rheumatoid factor in rheumatoid arthritis.

Rheumatoid arthritis (RA) is associated with higher levels of autoantibodies and IL-17. Here, we investigated if ectopic lymphoid follicles and peripheral blood mononuclear cells (PBMCs) from RA patients exhibit increased activation-induced cytidine deaminase (AID), and if increased AID is correlated with serum levels of autoantibodies and IL-17. The results of immunohistochemical staining showed that organized AID(+) germinal centres were observed in six of the 12 RA synovial samples, and AID(+) cells were found almost exclusively in the B-cell areas of these follicles. Aggregated but not organized lymphoid follicles were found in only one OA synovial sample without AID(+) cells. Significantly higher levels of AID mRNA (Aicda) detected by RT-PCR were found in the PBMCs from RA patients than PBMCs from normal controls (P < 0.01). In the PBMCs from RA patients, AID was expressed predominately by the CD10(+)IgM(+)CD20(+) B-cell population and the percentage of these cells that expressed AID was significantly higher than in normal controls (P < 0.01). AID expression in the PBMCs correlated significantly and positively with the serum levels of rheumatoid factor (RF) (P

Cigarette smoking, disease severity and autoantibody expression in African Americans with recent-onset rheumatoid arthritis.

OBJECTIVE: To examine the association of smoking with clinical and serological features in African Americans with recent-onset rheumatoid arthritis (RA) and to explore whether this association is dependent on the presence of the HLA-DRB1 shared epitope (SE). METHODS: In African Americans with recent-onset RA (n = 300), we examined the association of cigarette smoking (current versus past versus never and pack-years of exposure) with anti-cyclic citrullinated peptide antibody, rheumatoid factor (RF) (IgM and IgA), rheumatoid nodules and baseline radiographic erosions using logistic and cumulative logistic regression (adjusting for SE status). We also examined for evidence of interaction between smoking status and SE for all outcomes. RESULTS: Although there was no association with RF-IgA seropositivity, current smokers were approximately twice as likely as never smokers to have higher IgA-RF concentrations (based on tertiles; OR = 1.74; 95% CI 1.05 to 2.88) and nodules (OR = 2.43; 95% CI 1.13 to 5.22). These associations were most pronounced in those with more than 20 pack-years of exposure. There was no association of smoking status or cumulative tobacco exposure with anti-cyclic citrullinated peptide antibody, IgM-RF or radiographic erosions. There was also no evidence of a biological or statistical SE-smoking interaction for any of the outcomes examined. CONCLUSIONS: This is the first study to systematically examine the association of cigarette smoking with RA-related features in African Americans. Cigarette smoking is associated with both subcutaneous nodules and higher serum concentrations of IgA-RF in African Americans with RA, associations that may have important implications for long-term outcomes in this population.

Duration of preclinical rheumatoid arthritis-related autoantibody positivity increases in subjects with older age at time of disease diagnosis.

OBJECTIVES: To investigate factors that may influence the prevalence and timing of appearance of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibodies during the preclinical phase of rheumatoid arthritis (RA) development. METHODS: 243 serial prediagnosis serum samples from 83 subjects with RA were examined for the presence of RF and anti-CCP antibodies. RESULTS: Of the 83 cases, 47 (57%) and 51 (61%) subjects had at least one prediagnosis sample positive for RF or anti-CCP, respectively. Gender and race were not significantly associated with the prevalence or timing of preclinical antibody appearance. Preclinical anti-CCP positivity was strongly associated with the development of erosive RA (odds ratio = 4.64; 95% confidence interval 1.71 to 12.63; p<0.01), but RF was not (p = 0.60). Additionally, as age at the time of diagnosis of RA increased the duration of prediagnosis antibody positivity for RF and anti-CCP increased, with the longest duration of preclinical antibody positivity seen in patients diagnosed with RA over the age of 40. In no subjects did symptom onset precede the appearance of RF or anti-CCP antibodies. CONCLUSIONS: The period of time that RF and anti-CCP are present before diagnosis lengthens as the age at the time of diagnosis of RA increases. This finding suggests that factors such as genetic risk or environmental exposure influencing the temporal relationship between the development of RA-related autoantibodies and clinically apparent disease onset may differ with age.

Inhibition of complement component C3 reduces vein graft atherosclerosis in apolipoprotein E3-Leiden transgenic mice.

BACKGROUND: Venous bypass grafts may fail because of development of intimal hyperplasia and accelerated atherosclerosis. Inflammation plays a major role in these processes. Complement is an important part of the immune system and participates in the regulation of inflammation. The exact role of complement in the process of accelerated atherosclerosis of vein grafts has not yet been explored, however. METHODS AND RESULTS: To assess the role of complement in the development of vein graft atherosclerosis, a mouse model, in which a venous interposition was placed in the common carotid artery, was used. In this model, vein graft thickening appeared within 4 weeks. The expression of complement components was studied with the use of immunohistochemistry on sections of the thickened vein graft. C1q, C3, C9, and the regulatory proteins CD59 and complement receptor-related gene y could be detected in the lesions 4 weeks after surgery. Quantitative mRNA analysis for C1q, C3, CD59, and complement receptor-related gene y revealed expression of these molecules in the thickened vein graft, whereas C9 did not show local mRNA expression. Furthermore, interference with C3 activation with complement receptor-related gene y-Ig was associated with reduced vein graft thickening, reduced C3 and C9 deposition, and reduced inflammation as assessed by analysis of influx of inflammatory cells, such as leukocytes, T cells, and monocytes. In addition, changes in apoptosis and proliferation were observed. When C3 was inhibited by cobra venom factor, a similar reduction in vein graft thickening was observed. CONCLUSIONS: The complement cascade is involved in vein graft thickening and may be a target for therapy in vein graft failure disease.

Complement component c5a is integral to the febrile response of mice to lipopolysaccharide.

OBJECTIVES: The complement system is critical to the febrile response of mice to intraperitoneally administered lipopolysaccharide (LPS). We previously identified C3 and C5 as two components potentially involved in this response. This study was designed to examine whether the complement system is also pivotal in the response of mice to intravenously or intracerebroventricularly injected LPS, to distinguish between C3 and C5 and their cognate derivatives as the essential mediator(s), and to determine whether the failure of complement-deficient mice to develop a fever could be due to their possible inability to secrete pyrogenic cytokines. METHODS: Wild-type (WT; C57BL/6J) mice, hypocomplemented or not by intravenously injected cobra venom factor (10 U/mouse), and C3-, CR3- and C5-sufficient and -deficient mice were intravenously challenged with LPS (0.25 mug/mouse); WT and C3-/- mice pretreated with a C5a receptor antagonist (C5aRa) were similarly challenged. In addition, the serum levels of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and IL-6 were compared in LPS-treated C5+/+ and C5-/- mice. RESULTS: LPS induced a 1 degrees C rise in core temperature in all the mice, except C5-/- mice and those pretreated with C5aRa. C5+/+ and C5-/- mice challenged intracerebroventricularly with LPS exhibited identical febrile responses. LPS induced similar increases in the serum levels of IL-1beta, TNFalpha and IL-6 in C5+/+ and C5-/- mice. CONCLUSIONS: C5a is crucial for the development of febrile responses to LPS in mice; its site of action is peripheral, not central. The possibility that an inability to produce cytokines could account for the failure of C5-/- mice to develop a fever is not supported.

Association of rheumatoid arthritis treatment response and disease duration with declines in serum levels of IgM rheumatoid factor and anti-cyclic citrullinated peptide antibody.

OBJECTIVE: To examine the association of treatment response and disease duration with changes in rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibody levels among patients with rheumatoid arthritis (RA). METHODS: The study sample included 66 RA patients who completed double-blind, randomized clinical protocols and for whom baseline and followup serum samples were available. Anti-CCP and RF levels were measured using commercially available assay kits. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were used to describe the association of response and disease duration with declines in antibody levels. RESULTS: Patients had a mean +/- SD age of 49.9 +/- 12.0 years and were predominantly female (n = 51; 77%). The mean +/- SD duration between the times at which the baseline and followup serum samples were obtained was 13.7 +/- 8.6 months. Among the 64 subjects with positive antibody at baseline, 33 (52%) experienced a > or =25% reduction in the anti-CCP antibody level during the course of treatment, and 35 patients (55%) had a > or =25% reduction in RF. After adjustment for the baseline anti-CCP antibody level, only a shorter disease duration (< or =12 months) was significantly associated with a decline in the level of anti-CCP antibody (OR 3.0, 95% CI 1.0-8.8), and no association with treatment response was observed. Conversely, treatment response was the only significant determinant of a decrease in RF levels (OR 3.6, 95% CI 1.2-10.4). CONCLUSION: Shorter disease duration predicts greater declines in anti-CCP antibody levels with treatment in RA. Although treatment response is a robust determinant of a decrease in RF, it does not appear to be associated with declines in the anti-CCP antibody level.

Increased serum C3 levels in Crry transgenic mice partially abrogates its complement inhibitory effects.

Complement receptor 1-related gene/protein y (Crry) is a potent murine complement regulator that inhibits C3 convertases. Transgenic mice that overexpress soluble Crry (sCrry), directed systemically by the metallothionein-I promoter, have been used as an animal model for chronic blockade of complement activation. Recently we have found that alternative pathway (AP) activity in Crry transgenic mice was not inhibited as much as expected. To elucidate the mechanism of this effect, we evaluated the AP activities and levels of sCrry and AP complement components in transgenic and non-transgenic mice. In transgenic mice, expression of sCrry was induced by feeding zinc sulphate solution to 70.1 +/- 42.7 micro g/ml mean serum level. Its corresponding level of purified sCrry inhibited 49% of AP activity of normal mice serum; however, the actual AP activities in transgenic mice were not decreased when compared to non-transgenic mice (130.2 +/- 9.0%versus 113.0 +/- 35.4%). Expressed sCrry was functional, as immunoprecipitation and removal of sCrry from transgenic sera with rabbit anti-Crry polyclonal antibody resulted in enhanced AP activity, consistent with initial levels of sCrry. We then compared the changes to C3, factor B, factor H and factor D serum levels in transgenic and non-transgenic mice after induction of sCrry expression. Of these only C3 was increased after zinc feeding in transgenic mice compared to non-transgenic mice (142.8 +/- 14.1%versus 121.4 +/- 15.1%, P = 0.023). These results suggest that the inhibitory effect of chronic exposure to sCrry is compensated by concomitant alteration in C3 levels. This result also suggests the presence of a complement regulatory protein controls the level of serum C3, which has potential importance in the design and interpretation of studies involving chronic use of complement inhibitors.

Activation of complement mediates antiphospholipid antibody-induced pregnancy loss.

Although it is clear that the specific antigenic reactivity of antiphospholipid (aPL) antibodies is critical to their effect, the pathogenic mechanisms that result in injury in vivo are incompletely understood. We hyphothesized that aPL antibodies targeted to the placenta activate complement locally, generating split products that mediate placental injury and lead to foetal loss and growth retardation. To test this hypothesis, we used a murine model of APS in which pregnant mice are injected with human IgG containing aPL antibodies. Mice treated with inhibitors of complement activation and mice deficient in complement components were protected from aPL antibody-induced foetal damage. Although the cause of tissue injury in this disease is probably multifactoral, we have shown that complement activation is an absolute requirement for foetal loss and growth restriction and, therefore, thatthis pathway acts upstream of other important effector mechanisms. Identification of complement activation as a mechanism that is necessary for aPL-induced tissue damage and definition ofthe complement components necessary to trigger such injury is likely to lead to a better understanding of the pathogenesis of vascular and tissue injury in SLE and to new and improved treatments.

Structure of complement receptor (CR) 2 and CR2-C3d complexes.

Using X-ray crystallography, we have determined the structure of the first two short consensus repeats (SCRs) of human complement receptor (CR) 2 in complex with C3d. These studies revealed: (i) a primary site of interaction for C3d within SCR2 of CR2, (ii) a hydrophobic patch holding SCR1 to SCR2 in a rigid V-shape, (iii) a dimer formed by interactions between SCR1 of each molecule, (iv) several non-linear sequences on C3d that interact with CR2 and (v) mutations of C3d amino acids within the co-crystal interface that resulted in decreased binding. In addition, a polymorphism that results in decreased C3d binding and introduces a new glycosylation site predicted to disrupt the dimer interface was found in the New Zealand White autoimmune mouse strain. Although the co-crystal complex results are in agreement with a subset of prior studies, our additional findings, which demonstrate an extended SCR1-SCR2 structure in solution and differences in the kinetics of ligand-receptor interactions with longer forms of CR2, have suggested a more complex receptor-ligand interaction. To characterize this interaction further, several approaches directed at the determination of solution phase interactions as well as the analysis of the three-dimensional structure of CR2 alone and key CR2 mutants will be necessary.

Solution structures of complement components by X-ray and neutron scattering and analytical ultracentrifugation.

The short consensus/complement repeat (SCR) domain (also known as the complement control protein domain) is the most abundant domain type in the complement system. Crystal and NMR structures for proteins that contain single and multiple SCR domains have now been published. These contain inter-SCR linkers of between three and eight residues, and the structures show much variability in inter-SCR orientations. X-ray and neutron scattering, combined with analytical ultracentrifugation and constrained modelling based on known subunit structures will yield a medium-resolution structure for the protein of interest. The fewer parameters that are associated with the structure of interest, the more defined the structure of interest becomes. These solution studies have been applied to several SCR-containing proteins in the complement system, most notably Factor H with 20 SCR domains, a complement receptor type 2 fragment with two SCR domains, and rat complement receptor-related protein (Crry) which contains five SCR domains. The results show great conformational variability in the inter-SCR orientation, and these will be reviewed. Even though the rotational orientation cannot be modelled, it is nonetheless possible to measure the degree of extension of the multi-SCR proteins and, from this, to obtain functionally useful results.

Modulation of mouse endotoxic fever by complement.

It was recently reported that the complement system may be critically involved in the febrile response of guinea pigs to systemic, particularly intraperitoneally (i.p.) injected, lipopolysaccharides (LPS). The present study was designed to identify which component(s) of the complement cascade may be specifically critical. To this end, we used mice with C3, C5, and CR2 gene deletions. To assess preliminarily the suitability of mice for such a study, we replicated our earlier studies with guinea pigs. Thus, to verify initially whether complement is similarly involved in the febrile response of wild-type (C57BL/6J) mice to i.p. LPS (Escherichia coli, 1 microg/mouse), we depleted complement with cobra venom factor (CVF; 7 U/mouse, intravenously [i.v.]). These animals did not develop fever, whereas the core temperature (T(c)) of CVF vehicle-treated controls rose approximately 1 degrees C by 80 min postinjection and then gradually abated over the following 2.5 h, confirming the involvement of complement in fever production after i.p. LPS injection and the suitability of this species for these studies. C3- and C5-sufficient (C3(+/+) and C5(+/+)) mice also developed 1 degrees C fevers within 80 min after i.p. LPS (1 or 2 microg/mouse) injection. These fevers were totally prevented by CVF (10 U/mouse, i.v.) pretreatment. C3- and C5-deficient (C3(-/-) and C5(-/-)) mice were also unable to develop T(c) rises after i.p. LPS. Both CR2(+/+) and CR2(-/-) mice responded normally to i.p. LPS (1 microg/mouse). These data indicate that C5, but not C3d acting through CR2, may play a critical role in the febrile response of mice to i.p. LPS.